Growth of murine sarcoma virus-transformed rat kidney cells in nude mice: absence of induction of host endogenous viruses.

Clonal isolates of the normal rat kidney cell line (NRK) transformed by a defective murine sarcoma virus (Kirsten strain) were injected into nude mice of BALB/c background to determine whether the growth of these cells as tumors was accompanied by the induction of host endogenous type C viruses. All the virus-transformed clones produced rapidly growing tumors in nude mice, but neither the induction of mouse endogenous viruses nor the rescue and spread of the transforming sarcoma virus were observed during the growth of tumors. The degree of expression of the tumor virus structural proteins in the transformed cells did not determine the cellular phenotype with regard to tumorigenicity in nude mice, nor did it modify the cellular growth properties in vitro. Consistent with earlier observations with simian virus 40-transformed mouse and rat cells, the ability of sarcoma virus-transformed NRK cells to initiate tumor growth in nude mice appeared to be correlated with anchorage-independent growth in vitro.     

Transforming growth factor production by chemically transformed cells

Evidence is presented indicating that the chemically transformed AKR-MCA and C3H/MCA-58 murine cell lines produce "transforming growth factor(s)" capable of inducing a transformed morphology and the ability to grow in soft agar in nontransformed, anchorage-dependent indicator cells. Serum-free medium conditioned by exposure to the chemically transformed cells was chromatographed on a Bio-Gel P-60 column after dialysis and lyophilization. Using the nontransformed mouse AKR-2B cells as the indicator cells, a peak of soft agar growth-stimulating activity was detected in the molecular weight range of 10,000 to 12,000. The soft agar growth-stimulating activity in pooled fractions from the AKR-MCA cells was shown to be trypsin and dithiothreitol sensitive and relatively heat stable; the activity was not destroyed by heating to 56 degrees for 30 min or to 100 degrees for 3 min. The pooled material also caused stimulation of growth in the soft agar of rat NRK cells and stimulation of DNA synthesis in the AKR-2B cells. The quantity required to give significant competition for binding to the epidermal growth factor receptor was about one order of magnitude greater than that required for stimulation of soft agar growth. Further separation of these polypeptide(s) by carboxymethylcellulose chromatography revealed three apparent peaks of soft agar growth-stimulating activity. Epidermal growth factor receptor-competing activity cochromatographed with the early minor soft agar growth-stimulating peak, whereas the two major peaks of soft agar growth-stimulating activity had no associated detectable competition for epidermal growth factor binding to its receptor. The data indicate that at least a major portion of the transforming growth factors produced by the chemically transformed cells is different from those described previously in murine sarcoma virus-transformed mouse cells and human tumor cells.     

Normal Human Chromosome 1 Carries Suppressor Activity for Various Phenotypes of a Kirsten Murine Sarcoma Virus-transformed NIH/3T3 Cell Line

In order to identify chromosomes that carry putative tumor-suppressor genes for the various phenotypes of Kirsten sarcoma virus-transformed NIH/3T3 (DT) cells, we performed microcell-mediated chromosome transfer into DT cells. We first isolated mouse A9 clones, containing a single human chromosome 1, 11 or 12 tagged with pSV2- neo plasmid DNA. Then, chromosome 1, 11 or 12 was transferred from the A9 clones into DT cells by microcell fusion. The growth rate, colony-forming ability in soft agar and tumorigenicity of the DT cells were controlled by chromosome 1, but not by chromosome 11 or 12, indicating that normal human chromosome 1 carries a putative tumor-suppressor gene(s) that affects various transformed phenotypes of DT cells.     

Normal human chromosome 1 carries suppressor activity for various phenotypes of a Kirsten murine sarcoma virus-transformed NIH/3T3 cell line.

In order to identify chromosomes that carry putative tumor-suppressor genes for the various phenotypes of Kirsten sarcoma virus-transformed NIH/3T3 (DT) cells, we performed microcell-mediated chromosome transfer into DT cells. We first isolated mouse A9 clones, containing a single human chromosome 1, 11 or 12 tagged with pSV2-neo plasmid DNA. Then, chromosome 1, 11 or 12 was transferred from the A9 clones into DT cells by microcell fusion. The growth rate, colony-forming ability in soft agar and tumorigenicity of the DT cells were controlled by chromosome 1, but not by chromosome 11 or 12, indicating that normal human chromosome 1 carries a putative tumor-suppressor gene(s) that affects various transformed phenotypes of DT cells.     

Genetic stability of the sarcoma viruses in murine and avian sarcoma virus-transformed nonproducer cells

Clonal lines of murine and avion sarcoma virus-transformed mammalian cells, in which the viruses do not productively replicate, were studied for the occurrence of spontaneous or chemical-induced morphologic reversion. From over 10,000 single-cellderived clones of MSV-transformed nonproducer cells examined, only one morphologic revertant was detected. This cell line contained a normal murine sarcoma viral genome, suggesting that it arose as a result of mutation of a cellular gene involved in expression of the transformed state. The frequency of morphologic reversion of avian sarcoma virus-transformed mammalian cell lines was also investigated. The studies indicate that both murine and avian sarcoma viruses present in transformed nonproducer cells can exist in a genetically stable form.     

Separation of sarcoma growth inhibitor from avian sarcoma virus-transformed rat cells

A cell growth inhibitor was isolated from an avian sarcoma virus-transformed rat cell line (77N1), from which we had previously obtained a novel transforming growth factor, TGF gamma 2. The growth inhibitor (named SGI) was found in cell extract of 77N1 cells and was separable from TGF gamma 2 by means of ion exchange chromatography. SGI inhibited the DNA synthesis of serum-starved, TGF gamma 2-stimulated BALB3T3 cells as well as the spontaneous and TGF-induced colony formation of various cells in semisolid agar. We present evidence that SGI is distinct from the ubiquitous growth modulator TGF beta, and we discuss the possible role of SGI in the neoplastic cell proliferation.     

Anchorage-independent growth-conferring factor production by rat mammary tumor cells

Conditioned medium from cultures of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells contain factors that resemble sarcoma growth factor and other transforming growth factors in biological activity but differ in their physical properties. The mammary tumor factors (MTF) are acid stable and heat and protease sensitive. They inhibit the binding of epidermal growth factor, but not insulin, to mouse embryonal carcinoma cells. MTF confers upon normal rat kidney and BALB/c-3T3 cells the ability to grow in soft agar. This effect is enhanced synergistically by high concentrations of fetal calf serum but not by epidermal growth factor. Anchorage-independent growth promotion, however, is not seen with normal mammary epithelial cells, although MTF is mitogenic for these cells as well as normal rat kidney cells, BALB/c-3T3 cells, and chick embryo fibroblasts in monolayer culture, MTF is not mitogenic for primary cultures of the tumor cells from which the factors are derived. Two major molecular weight species of MTF, eluting at Mr 6,000 and 65,000 to 70,000 on Bio-Gel P-100 columns, are present in acid-ethanol extracts of 7,12-dimethylbenz(a)anthracene- and nitrosomethylurea-induced rat mammary tumors. Transplantable tumors derived from primary 7,12-dimethylbenz(a)anthracene- or nitrosomethylurea-induced tumors have little or no MTF activity. These results demonstrate that different chemically induced rat mammary tumors contain transforming growth factor-like activities. Furthermore, it is possible that MTF is unnecessary for the maintenance of tumorigenicity, since some tumors contain no detectable MTF.     

Specific antigrowth effect of interferon on mouse cells transformed by murine sarcoma virus

The growth rate of NIH/3T3 mouse fibroblasts transformed by the Moloney strain of murine sarcoma virus was investigated following interferon (IFN) treatment. These cells were found to be sensitive to the antigrowth effect of IFN as indicated by a slower growth rate in its presence. The effect was most efficiently expressed when cells were grown at low serum concentrations, e.g., 2.5%. Likewise, IFN treatment caused a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. In contrast, these effects were observed only to a very minor extent with uninfected NIH/3T3 cells or with NIH/3T3 cells chronically infected with murine leukemia virus. In addition, IFN treatment significantly decreased the cloning efficiency of murine sarcoma virus-transformed cells in semi-solid agar. Furthermore, an even stronger effect on the cloning efficiency was observed in liquid medium supplied with 2.5% serum, indicating a direct inhibitory effect on the growth of these cells. Under these conditions, NIH/3T3 or NIH/3T3(MLV) cells remained unaffected by IFN treatment, nor was this effect evident when the transformed cells were grown in the presence of 10% serum. A 4-fold increase in the level of (2'-5')oligoadenylate synthetase following IFN treatment was observed in murine sarcoma virus-transformed cells as compared to either NIH/3T3 or NIH/3T3(MLV) cells.     

Altered glucose transport kinetics in murine sarcoma virus-transformed BALB-3T3 clones

The kinetics of glucose transport were determined for a number of spontaneous and virus-transformed cell lines derived from a single clone of BALB/3T3 cells. SV40-transformed and mouse leukemia virus-infected cells were characterized by a Km very similar to that of the parental cell line. In contrast, murine sarcoma virus-transformed cells had a much lower Km. These findings are in agreement with previous results obtained with mass cultures of NIH Swiss mouse embryo cells. One X-irradiation and two spontaneous transformants also showed low Km values and no virions or virion antigens. These latter cell lines should provide an opportunity for a crucial test of the relationship between glucose transport kinetics and a functional sarcoma virus genome.     

Presence of heparin binding growth factor in mouse bladder tumors and urine from mice with bladder cancer

Heparin affinity chromatography has been used to partially purify angiogenic factors from normal and neoplastic tissue. The same technique was used to partially purify angiogenic-like factors from two mouse bladder tumors and urine from mice with bladder cancer. Both MBT-2 and MB49 tumors contained heparin-binding 3T3 cell growth factor activity that was eluted by 1.2 to 1.4 M salt. The growth factor isolated from MBT-2 tumor was mitogenic for capillary endothelial cells. Analysis of the 1.2 M heparin eluate by high-pressure liquid chromatography showed that it consisted of two 3T3 cell growth factors with molecular weights of 16,000 and 26,000. The growth factor activity isolated from MB49 tumors had an affinity for Bio-rex 70 which was similar to other cationic heparin binding growth factors. Analysis of urine pooled from tumor-bearing mice by heparin-Sepharose chromatography demonstrated 3T3 cell growth factor activity in fractions eluted with 1 to 1.4 and 2.5 M dsalt, whereas no significant growth factor activity was detected in pooled urine from control mice. The growth factor activity found in mouse bladder tumors differed from epidermal growth factor, transforming growth factor-alpha, and platelet-derived growth factor in terms of affinity for heparin-Sepharose and molecular weight. The observation that urine from tumor-bearing mice contains increased concentrations of this growth factor compared to normal urine suggests that a similar relationship may exist for human urine.     

No expression of a Rous sarcoma virus-induced tumor antigen in mammalian cells infected with retroviruses transducing other oncogenes of the src gene family

Immunization with mouse and rat cells transformed by Rous sarcoma virus (RSV) or by B77 avian sarcoma virus (ASV) induced complete transplantation resistance against an RSV-induced mouse tumor (CSA1M) in syngeneic hosts. In contrast, most of the mice immunized with a Fujinami sarcoma virus-transformed rat fibroblast line (FSV-3Y1), a feline sarcoma virus-transformed cat fibroblast line (FeSV-FEF), an Abelson leukemia virus-infected Balb/3T3 cell line (AbLV-3T3), or an uninfected 3Y1 cell line could not reject the CSA1M. Serologic analysis with the use of a complement-dependent cytotoxicity assay supported the results of transplantation studies. The mouse and rat cells transformed by RSV or B77 ASV expressed a common tumor-specific cell surface antigen (TSSA) detected by syngeneic antiserum against the CSA1M, whereas none of the FSV-3Y1, FeSV-FEF, and AbLV-3T3 cells expressed the TSSA. These results suggest that the common TSSA in the mouse and rat cells transformed by RSV or B77 ASV containing src gene is not shared with mammalian cells infected with retroviruses transducing other oncogenes of the src gene family (i.e., fps, fes, and abl).     

Detection of a transforming gene in spontaneous reticulum cell sarcoma of SJL/J mice: genetically linked and host-dependent neoplasia

Spontaneous reticulum cell sarcoma (RCS) tumor induction occurs in 90% of SJL/J mice of 8-13 months of age. Tumor induction and growth has been shown to be under the influence of both H-2 and non-H-2 genes as well as the presence of an intact host T-cell system. We postulated that cellular oncogenes may play a role in the induction, growth, and characteristics of RCS. DNA-mediated gene transfer protocols were adopted to investigate the presence of transforming genes in DNA from RCS of SJL/J mice. High molecular weight DNA was isolated from these tumors as well as from brains and livers of control tumor-free SJL/J mice and transfected into NIH-3T3 mouse and F2408 rat fibroblast cell lines. Foci of transformed cells with a peculiar round morphology were scored in both rat and mouse cultures given tumor DNA, but not in those receiving DNA from normal tissues. DNA from first-cycle transformants was transfected in further cycles of transfection, giving rise to foci with similar morphological appearances and growth properties. These experiments suggest that a transforming gene, present in RCS spontaneous tumors, is involved in the malignant conversion of the transfected normal fibroblasts. The implication of these results with respect to the induction and growth properties of RCS is discussed.     

Synergistic interaction of two classes of transforming growth factors from murine sarcoma cells

Transforming growth factors (TGFs) isolated from murine sarcoma virus-transformed 3T3 cells have been separated by high-pressure liquid chromatography into two subsets. One subset, called TGF alpha, competes with epidermal growth factor (EGF) for receptor sites, whereas the other, called TGF beta, does not. TGB beta, purified by high-pressure liquid chromatography, will not induce formation of large colonies of cells in soft agar in the absence of TGF alpha or EGF. However, the combined action of either TGF alpha or EGF (which by themselves are relatively ineffective in promoting growth of cells in soft agar) together with TGF beta results in a potent synergistic effect, with formation of large colonies. Chemically modified analogs of EGF also potentiate TGF beta activity to the extent that they bind to the EGF receptor. It is suggested that TGF beta may be an important mediator of the known effects of both TGF alpha and EGF on neoplastic transformation.     

Changes in leucine aminotransferase isozymes by viral transformation and its correlation with the isozyme changes occurring during differentiation.

The isozyme pattern of leucine aminotransferase (EC 2.6.1.6) in various cell lines and their viral-transformed derivatives were examined. The Wistar 3C rat liver cell line was found to contain only isozyme I, while its simian virus 40-transformed counterpart had isozyme III in addition to isozyme I. A spontaneously transformed late passage clone of these liver cells was also found to have acquired isozyme III. Polyoma virus-transformed baby hamster kidney cells were also found to contain a greater predominance of isozyme III than their normal untransformed counterpart. Examination of the isozymes in a cloned normal rat kidney cell line transformed by a mutant of Rous sarcoma virus which is temeprature-sensitive for transformation indicated that, in fact, such an isozyme change does correlate with transformation. When grown at 36 degrees these cells contained predominantly isozyme III; however, upon reacquiring normal morphology and lowered glucose transport activity when grown at 40 degrees, their isozyme pattern was now found to be changed and consisted predominantly of isozyme I, as is found in normal adult rat kidney tissue. Isozyme III was found to be present in neonatal kidney tissue of the rat and hamster, and its predominance in the virus-transformed normal rat and baby hamster kidney cells was interpreted as indicative of the dedifferentiation of these cells upon viral transformation. A similar change of the isozyme pattern of leucine aminotransferase in chicken embryos during their development was observed, such that in 5-day-old embryos Form III was predominant, while in the more mature differentiated chicken embryo of Day 17, Form I was predominant.     

Pericellular matrix and cell surface glycoproteins of virus-transformed mouse epithelial cells

Mouse embryo Mus musculus castaneous epithelial cells, transformed with Moloney murine sarcoma virus (MSV) or with ecotropic murine leukemia virus (MuLV), were analyzed for production of pericellular matrix glycoproteins. The nontransformed, MSV-transformed, and MuLV-transformed cells produced fibronectin, laminin, type I collagen, and small amounts of type III collagen when studied by immunofluorescence using specific antibodies. The virus-transformed epithelial cells produced enhanced amounts of fibronectin into their growth media. Nontransformed M. musculus castaneous epithelial cells mainly produced type I collagen, as shown by metabolic labeling and polypeptide analysis. A significant increase in the glycoprotein production was seen by the MuLV-transformed cells, whereas small changes in the collagen production were apparent after MSV transformation. MuLV-transformed cells produced increased amounts of type I collagen and also some collagenous polypeptides that comigrated with procollagen type IV chains. The ratio of the procollagen type I chains deposited in the matrix was altered in transformed cells. Radioactive surface labeling of the cells revealed changes of the high-molecular-weight glycoproteins in both the MSV- and the MuLV-transformed cells. Unlike virus-transformed fibroblastic cells, these transformed epithelial cells deposited and retained connective tissue glycoproteins in their pericellular matrices. The results indicate that viral transformation modulates the pericellular matrix and surface glycoproteins of cultured mouse epithelial cels. The ability of virus-transformed epithelial cells to deposit pericellular matrices is a major difference between them and virus-transformed fibroblastic cells.     

Isolation of tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line

Two types of growth-modulating factors were derived from the serum-free conditioned media of a human rhabdomyosarcoma cell line, A673. One type, Mr 18,000 to 22,000, competes for binding to epidermal growth factor receptors and enhances the growth of normal and tumor cells in soft agar. It has all of the biological properties ascribed to transforming growth factor type alpha (TGF alpha). A673 cells also produce factors which inhibit the growth of human tumor cells in soft agar and in monolayer cultures. These tumor cell growth-inhibiting factors (TIFs) are acid- and heat-stable peptides. The major TIF activities have molecular weights in the ranges of greater than 28,000, 18,000 to 22,000, 10,000 to 16,000, and 5,000 to 10,000 and do not possess the antiviral activity associated with interferon. Partially purified preparations of TIF-1 (Mr 10,000 to 16,000) inhibit the growth of all human tumor cell lines tested and stimulate the growth of normal human fibroblasts and epithelial cells in monolayer cultures. The growth of human lung carcinoma A549 cells in soft agar, which was enhanced by treatment with TGF alpha from A673-conditioned media, was inhibited by treatment with TIF-1 derived from the same media. The ratio of the two types of tumor cell-derived, growth-modulating factors (TIFs and TGF alpha), which are antagonistic in their biological effects, may determine the capacity of tumor cells for anchorage-independent growth.     

Damaging action of human recombinant TNF on tumor vessels as an aspect of its anti-neoplastic action against Meth A sarcoma in mice

Effect of human recombinant TNF (rHu-TNF) on tumor blood vessels was investigated in relation to its mode of anti-neoplastic action against Meth A sarcoma in BALB/c mice. The extent of the blood vessel lesion was evaluated by measuring the degrees of blueing and hemorrhage after intravenous injection of Evan's blue. When injected intravenously into mice bearing Meth A cells in the abdominal skin transplanted 8 days previously, rHu-TNF, at doses of 3000 and 10,000 units/mouse produced dose-related lesioning of blood vessels in the sarcoma and the adjacent skin but not in the skin distant from the sarcoma. Similar lesioning of blood vessels in the adjacent skin was seen after intravenous administration of 10,000 units/mouse of rHu-TNF in mice with the sarcoma in which the cells had been selectively killed by intratumoral injection of acetic acid (20%, 0.01 ml). On the other hand, rHu-TNF (10,000 units/mouse, i.v.) produced weak lesioning of blood vessels in the granuloma tissue caused by subcutaneous implantation of felt pellets and in the adjacent skin but not in the operation wound in mice with the sarcoma. These results suggest that rHu-TNF damages newly formed blood vessels, particularly those connected with the sarcoma, without influencing blood vessels in normal skin and healing wounds in mice with Meth A sarcoma.     

Cells derived from sporadic and AIDS-related Kaposi's sarcoma reveal identical cytochemical and molecular properties in vitro

We have established 40 cell cultures from biopsies of 5 patients with sporadic Kaposi's sarcoma (KS) and compared them with cell cultures derived from KS biopsies of AIDS patients. Immunocytochemical staining of sporadic KS cells revealed the same endothelial cell markers as those expressed on AIDS-KS cells, indicating that both types of KS might be of endothelial cell origin. In contrast to clinical features, in vitro growth properties showed no differences. KS cells of both types reveal a low degree of malignancy but can be distinguished from fibroblasts by a higher passage number in low serum concentrations and a more pronounced dependence on platelet-derived growth factor (PDGF). The DNA of 12 cell lines of both types of KS was negative for genomic equivalents of hepatitis B virus (HBV), cytomegalovirus (CMV) and, in the case of AIDS-KS, for human immunodeficiency virus (HIV). No rearrangements or amplifications of several oncogenes, often involved in human tumors, were detected. The expression of these oncogenes by sporadic and AIDS-KS cells, as analyzed by Northern blotting, was comparable to that of normal dermal fibroblasts from the same patients. Our results indicate that the 2 types of Kaposi's sarcoma possess identical cytochemical and molecular properties. They are probably weakly malignant neoplasms of endothelial cell origin, and paracrine growth stimulation appears to be important for their maintenance and progression.     

Immortalized mouse fetal liver stromal cells support growth and maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by co-culture with feeder cells. The feeder cells are important for the growth of hESCs. A novel spontaneously immortalizated mouse fetal liver stromal cell line, named KM3, was isolated from a?13.5?day mouse fetal liver. In this study, we examined whether KM3 cells could be used as feeders to support the growth of hESCs. hESCs cultured on KM3 cells showed a similar proliferation rate and characteristics to mouse embryonic fibroblasts?(MEFs) after prolonged culture, including morphology, unlimited and undifferentiated proliferative ability, maintenance of normal karyotypes, formation of embryoid bodies in?vitro and typically immature teratomas in?vivo. Our results indicate that the immortalized KM3 cell line has the potential to support the growth and maintenance of hESCs. The cell line may be used for the large-scale expansion of hESCs in a low-cost and less labor-intensive manner.     

Platelet-derived growth factor: roles in normal and v-sis transformed cells

Platelet-derived growth factor (PDGF) is the most potent mitogenic protein in human serum. The normal roles of this protein may relate to its potent mitogenic properties and its activities in directed cell migration and at sites of wounds. PDGF is a heterodimeric protein of approximately 30,000 molecular weight; one polypeptide chain of PDGF is highly homologous to the predicted amino acid sequence of p28v-sis, the putative transforming protein of simian sarcoma virus (SSV), suggesting a major role of growth factor activity in transformation by SSV. PDGF-like growth promoting activity is found in SSV-transformed cells and is secreted into conditioned media where it appears to interact with PDGF cell surface receptors to stimulate 3H-thymidine incorporation into DNA of cells secreting this protein. Transformation by SSV may in part be mediated by the autocrine stimulation of cell growth by the PDGF-like mitogenic properties of the transforming protein of SSV.